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- 详细信息
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pK7GWIWG2(I)
- 库存：
100
- 供应商：
上海烜雅生物科技有限公司
- 规格：
干粉/液体
基本信息
名称：pK7GWIWG2(I)植物表达质粒
别称: pK7GWIWG2(I)
质粒属性
质粒简介
pK7GWIWG2(I)是一个植物表达质粒。
质粒图谱
质粒序列
LOCUS Exported 13599 bp ds-DNA circular SYN 18-JUN-2017
DEFINITION synthetic circular DNA
KEYWORDS pK7GWIWG2(I)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13599)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..13599
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 28..253
/label=35s Terminator
protein_bind 263..387
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter 412..442
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
CDS 496..1155
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 1497..1802
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLL-SDK
VSRELYPVVHIGDESWRMMTTD-MASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(1843..1967)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
misc_feature complement(2035..2678)
/label=intron
protein_bind 2693..2817
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
CDS complement(2858..3163)
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLS-DK
VSRELYPVVHIGDESWRMMTTDM-ASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
CDS complement(3505..4164)
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
promoter complement(4218..4248)
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
protein_bind complement(4273..4397)
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter complement(4425..5460)
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
misc_feature 5482..5681
/label=RB
misc_feature 5543..5567
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 6867..7496
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 StaA
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 7930..8997
/codon_start=1
/product="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 RepA
/translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
MRYRNLIEGEASAGS"
rep_origin 9063..9257
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 9601..9741
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(9927..10515)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(10759..11550)
/codon_start=1
/gene="aadA"
/product="aminoglycoside adenylyltransferase (Murphy,
1985)"
/label=Spe
/note="confers resistance to spectinomycin and
streptomycin"
/translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
misc_feature 11840..12172
/label=LB
misc_feature 12078..12102
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
terminator 12195..12447
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(12487..13281)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(13315..13494)
/label=NOS promoter
/note="nopaline synthase promoter"
ORIGIN
1 ctagagggcc cgacgtcgca tgcctgcagg tcactggatt ttggttttag gaattagaaa
61 ttttattgat agaagtattt tacaaataca aatacatact aagggtttct tatatgctca
121 acacatgagc gaaaccctat aagaacccta attcccttat ctgggaacta ctcacacatt
181 attctggaga aaaatagaga gagatagatt tgtagagaga gactggtgat ttttgcggac
241 tctagcatgg ccgcgggata tcacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa
301 tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta cataatactg
361 taaaacacaa catatccagt cactatggcg gccgcattag gcaccccagg ctttacactt
421 tatgcttccg gctcgtataa tgtgtggatt ttgagttagg atccggcgag attttcagga
481 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa
541 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag
601 accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt
661 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg
721 gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc
781 catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag
841 tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct
901 aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt
961 tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa
1021 tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtc
1081 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg
1141 cagggcgggg cgtaaacgcg tggatccggc ttactaaaag ccagataaca gtatgcgtat
1201 ttgcgcgctg atttttgcgg tataagaata tatactgata tgtatacccg aagtatgtca
1261 aaaagaggtg tgctatgaag cagcgtatta cagtgacagt tgacagcgac agctatcagt
1321 tgctcaaggc atatatgatg tcaatatctc cggtctggta agcacaacca tgcagaatga
1381 agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat caggaaggga tggctgaggt
1441 cgcccggttt attgaaatga acggctcttt tgctgacgag aacagggact ggtgaaatgc
1501 agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga
1561 gtgatattat tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc
1621 tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc
1681 gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg
1741 atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat
1801 aaatgtcagg ctcccttata cacagccagt ctgcaggtcg accatagtga ctggatatgt
1861 tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata
1921 tttatatcat tttacgtttc tcgttcagct ttcttgtaca aagtggtgat atcactagtg
1981 cggccgcctg caggtcgacc atatggtcga cctgcaggcg gccgcactag tgatgctgtt
2041 atgttcagtg tcaagctgac ctgcaaacac gttaaatgct aagaagttag aatatatgag
2101 acacgttaac tggtatatga ataagctgta aataaccgag tataaactca ttaactaata
2161 tcacctctag agtataatat aatcaaattc gacaatttga ctttcaagag taggctaatg
2221 taaaatcttt atatatttct acaatgttca aagaaacagt tgcatctaaa cccctatggc
2281 catcaaattc aatgaacgct aagcttaata tgactctcaa taaagtctca taccaacaag
2341 tgccacctta ttcaaccatc aagaaaaaag ccaaaattta tgctactcta aggaaaactt
2401 cactaaagaa gacgatttag agtgttttac caagaatttc tgtcatctta ctaaacaact
2461 aaagatcggt gtgatacaaa acctaatctc attaaagttt atgctaaaat aagcataatt
2521 ttacccacta agcgtgacca gataaacata actcagcaca ccagagcata tatattggtg
2581 gctcaaatca tagaaactta cagtgaagac acagaaagcc gtaagaagag gcaagagtat
2641 gaaaccttac ctcatcattt ccatgaggtt gcttctgatc ccgcgggata tcaccacttt
2701 gtacaagaaa gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat
2761 tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt cactatggtc
2821 gacctgcaga ctggctgtgt ataagggagc ctgacattta tattccccag aacatcaggt
2881 taatggcgtt tttgatgtca ttttcgcggt ggctgagatc agccacttct tccccgataa
2941 cggagaccgg cacactggcc atatcggtgg tcatcatgcg ccagctttca tccccgatat
3001 gcaccaccgg gtaaagttca cgggagactt tatctgacag cagacgtgca ctggccaggg
3061 ggatcaccat ccgtcgcccg ggcgtgtcaa taatatcact ctgtacatcc acaaacagac
3121 gataacggct ctctctttta taggtgtaaa ccttaaactg catttcacca gtccctgttc
3181 tcgtcagcaa aagagccgtt catttcaata aaccgggcga cctcagccat cccttcctga
3241 ttttccgctt tccagcgttc ggcacgcaga cgacgggctt cattctgcat ggttgtgctt
3301 accagaccgg agatattgac atcatatatg ccttgagcaa ctgatagctg tcgctgtcaa
3361 ctgtcactgt aatacgctgc ttcatagcac acctcttttt gacatacttc gggtatacat
3421 atcagtatat attcttatac cgcaaaaatc agcgcgcaaa tacgcatact gttatctggc
3481 ttttagtaag ccggatccac gcgtttacgc cccgccctgc cactcatcgc agtactgttg
3541 taattcatta agcattctgc cgacatggaa gccatcacag acggcatgat gaacctgaat
3601 cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg
3661 ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg
3721 attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc
3781 accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta
3841 ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg
3901 aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc
3961 attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt
4021 tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc
4081 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
4141 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgccggatc
4201 ctaactcaaa atccacacat tatacgagcc ggaagcataa agtgtaaagc ctggggtgcc
4261 taatgcggcc gccatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt
4321 ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcagct
4381 tttttgtaca aacttgtgat atcactagtg cggccgcctg caggtcgact agaatagtaa
4441 attgtaatgt tgtttgttgt ttgttttgtt gtggtaattg ttgtaaaaat acggatcgtc
4501 ctgcagtcct ctccaaatga aatgaacttc cttatataga ggaagggtct tgcgaaggat
4561 agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg
4621 aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt
4681 tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc
4741 atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc
4801 aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt
4861 ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca
4921 tgttgacgaa gattttcttc ttgtcattga gtcgtaaaag actctgtatg aactgttcgc
4981 cagtcttcac ggcgagttct gttagatcct cgatctgaat ttttgactcc atggcctttg
5041 attcagtagg aactactttc ttagagactc caatctctat tacttgcctt ggtttatgaa
5101 gcaagccttg aatcgtccat actggaatag tacttctgat cttgagaaat atatctttct
5161 ctgtgttctt gatgcagtta gtcctgaatc ttttgactgc atctttaacc ttcttgggaa
5221 ggtatttgat ctcctggaga ttattactcg ggtagatcgt cttgatgaga cctgccgcgt
5281 aggcctctct aaccatctgt gggtcagcat tctttctgaa attgaagagg ctaatcttct
5341 cattatcggt ggtgaacatg gtatcgtcac cttctccgtc gaactttctt cctagatcgt
5401 agagatagag aaagtcgtcc atggtgatct ccggggcaaa ggagatcagc ttggctctag
5461 tcgaccatat gggagagctc aagcttagct tgagcttgga tcagattgtc gtttcccgcc
5521 ttcagtttaa actatcagtg tttgacagga tatattggcg ggtaaaccta agagaaaaga
5581 gcgtttatta gaataacgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt
5641 tgtatgtgca tgccaaccac agggttcccc tcgggatcaa agtactttga tccaacccct
5701 ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag ccgtcttctg aaaacgacat
5761 gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg cccttttcct ggcgttttct
5821 tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac tagaaccgga gacattacgc
5881 catgaacaag agcgccgccg ctggcctgct gggctatgcc cgcgtcagca ccgacgacca
5941 ggacttgacc aaccaacggg ccgaactgca cgcggccggc tgcaccaagc tgttttccga
6001 gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc aggatgcttg accacctacg
6061 ccctggcgac gttgtgacag tgaccaggct agaccgcctg gcccgcagca cccgcgacct
6121 actggacatt gccgagcgca tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc
6181 gtgggccgac accaccacgc cggccggccg catggtgttg accgtgttcg ccggcattgc
6241 cgagttcgag cgttccctaa tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc
6301 ccgaggcgtg aagtttggcc cccgccctac cctcaccccg gcacagatcg cgcacgcccg
6361 cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca
6421 tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa gtgacgccca ccgaggccag
6481 gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc gacgccctgg cggccgccga
6541 gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg acggccagga cgaaccgttt
6601 ttcattaccg aagagatcga ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc
6661 gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg gtttgtctga tgccaagctg
6721 gcggcctggc cggccagctt ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga
6781 tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta
6841 aataaacaaa tacgcaaggg gaacgcatga aggttatcgc tgtacttaac cagaaaggcg
6901 ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc cctgcaactc gccggggccg
6961 atgttctgtt agtcgattcc gatccccagg gcagtgcccg cgattgggcg gccgtgcggg
7021 aagatcaacc gctaaccgtt gtcggcatcg accgcccgac gattgaccgc gacgtgaagg
7081 ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc ccaggcggcg gacttggctg
7141 tgtccgcgat caaggcagcc gacttcgtgc tgattccggt gcagccaagc ccttacgaca
7201 tatgggccac cgccgacctg gtggagctgg ttaagcagcg cattgaggtc acggatggaa
7261 ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg
7321 ttgccgaggc gctggccggg tacgagctgc ccattcttga gtcccgtatc acgcagcgcg
7381 tgagctaccc aggcactgcc gccgccggca caaccgttct tgaatcagaa cccgagggcg
7441 acgctgcccg cgaggtccag gcgctggccg ctgaaattaa atcaaaactc atttgagtta
7501 atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc
7561 acgcagcagc aaggctgcaa cgttggccag cctggcagac acgccagcca tgaagcgggt
7621 caactttcag ttgccggcgg aggatcacac caagctgaag atgtacgcgg tacgccaagg
7681 caagaccatt accgagctgc tatctgaata catcgcgcag ctaccagagt aaatgagcaa
7741 atgaataaat gagtagatga attttagcgg ctaaaggagg cggcatggaa aatcaagaac
7801 aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg aacgggcggt tggccaggcg
7861 taagcggctg ggttgtctgc cggccctgca atggcactgg aacccccaag cccgaggaat
7921 cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg gcgcggcgct gggtgatgac
7981 ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca
8041 cgccccggtg aatcgtggca agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg
8101 ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg gcgacgagca accagatttt
8161 ttcgttccga tgctctatga cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc
8221 gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg tgatccgcta cgagcttcca
8281 gacgggcacg tagaggtttc cgcagggccg gccggcatgg ccagtgtgtg ggattacgac
8341 ctggtactga tggcggtttc ccatctaacc gaatccatga accgataccg ggaagggaag
8401 ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg acgtactcaa gttctgccgg
8461 cgagccgatg gcggaaagca gaaagacgac ctggtagaaa cctgcattcg gttaaacacc
8521 acgcacgttg ccatgcagcg tacgaagaag gccaagaacg gccgcctggt gacggtatcc
8581 gagggtgaag ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag
8641 tacatcgaga tcgagctagc tgattggatg taccgcgaga tcacagaagg caagaacccg
8701 gacgtgctga cggttcaccc cgattacttt ttgatcgatc ccggcatcgg ccgttttctc
8761 taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca gatggttgtt caagacgatc
8821 tacgaacgca gtggcagcgc cggagagttc aagaagttct gtttcaccgt gcgcaagctg
8881 atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg aggcggggca ggctggcccg
8941 atcctagtca tgcgctaccg caacctgatc gagggcgaag catccgccgg ttcctaatgt
9001 acggagcaga tgctagggca aattgcccta gcaggggaaa aaggtcgaaa aggtctcttt
9061 cctgtggata gcacgtacat tgggaaccca aagccgtaca ttgggaaccg gaacccgtac
9121 attgggaacc caaagccgta cattgggaac cggtcacaca tgtaagtgac tgatataaaa
9181 gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc
9241 cgcctggcct gtgcataact gtctggccag cgcacagccg aagagctgca aaaagcgcct
9301 acccttcggt cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat cgcggccgct
9361 ggccgctcaa aaatggctgg cctacggcca ggcaatctac cagggcgcgg acaagccgcg
9421 ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga
9481 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
9541 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
9601 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
9661 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
9721 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
9781 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
9841 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
9901 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
9961 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
10021 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
10081 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
10141 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
10201 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
10261 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
10321 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
10381 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
10441 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
10501 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
10561 gaaaactcac gttaagggat tttggtcatg catgatatat ctcccaattt gtgtagggct
10621 tattatgcac gcttaaaaat aataaaagca gacttgacct gatagtttgg ctgtgagcaa
10681 ttatgtgctt agtgcatcta atcgcttgag ttaacgccgg cgaagcggcg tcggcttgaa
10741 cgaatttcta gctagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg
10801 gacaaattct tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata
10861 agcctgtcta gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca
10921 gtcggcagcg acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga
10981 caacgtaagc actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt
11041 taaggtttca tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc
11101 cgccgctgga cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag
11161 atcaatgtcg atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc
11221 tccaaattgc agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac
11281 aatggtgact tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa
11341 aaggtcgttg atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag
11401 caaatcaata tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac
11461 ggccagcaac gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt
11521 cgatacttcg gcgatcaccg cttcccccat gatgtttaac tttgttttag ggcgactgcc
11581 ctgctgcgta acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc
11641 ttgctgcttg gatgcccgag gcatagactg taccccaaaa aaacatgtca taacaagaag
11701 ccatgaaaac cgccactgcg ccgttaccac cgctgcgttc ggtcaaggtt ctggaccagt
11761 tgcgtgacgg cagttacgct acttgcatta cagcttacga accgaacgag gcttatgtcc
11821 actgggttcg tgcccgaatt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca
11881 tgctaccctc cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg
11941 aatagcatcg gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg
12001 tcccggactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg
12061 agctgttggc tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa
12121 cttaataaca cattgcggac gtttttaatg tactgaatta acgccgaatt gaattatcag
12181 cttgcatgcc ggtcgatcta gtaacataga tgacaccgcg cgcgataatt tatcctagtt
12241 tgcgcgctat attttgtttt ctatcgcgta ttaaatgtat aattgcggga ctctaatcat
12301 aaaaacccat ctcataaata acgtcatgca ttacatgtta attattacat gcttaacgta
12361 attcaacaga aattatatga taatcatcgc aagaccggca acaggattca atcttaagaa
12421 actttattgc caaatgtttg aacgatctgc ttgactctag ctagagtccg aaccccagag
12481 tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc
12541 ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat
12601 atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc
12661 gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccgtg
12721 ggtcacgacg agatcctcgc cgtcgggcat ccgcgccttg agcctggcga acagttcggc
12781 tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat
12841 ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg
12901 atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc
12961 aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc
13021 cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga
13081 tagccgcgct gcctcgtctt ggagttcatt cagggcaccg gacaggtcgg tcttgacaaa
13141 aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt
13201 ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg
13261 caatccatct tgttcaatca tgcctcgatc gagttgagag tgaatatgag actctaattg
13321 gataccgagg ggaatttatg gaacgtcagt ggagcatttt tgacaagaaa tatttgctag
13381 ctgatagtga ccttaggcga cttttgaacg cgcaataatg gtttctgacg tatgtgctta
13441 gctcattaaa ctccagaaac ccgcggctga gtggctcctt caacgttgcg gttctgtcag
13501 ttccaaacgt aaaacggctt gtcccgcgtc atcggcgggg gtcataacgt gactccctta
13561 attctcatgt atgataattc gagggtaccc ggggatcct
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
名称：pK7GWIWG2(I)植物表达质粒
别称: pK7GWIWG2(I)
| 复制子: | pUC |
|---|---|
| 原核抗性: | Chl |
| 筛选标记: | G418 |
| 克隆菌株: | DB3.1 |
| 培养条件: | 37度 |
质粒属性
| 载体宿主： | 植物 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | |
| 基因类型： | GateWay |
| 原核抗性： | Chl |
| 筛选标记： | Neo/G418 |
| 荧光蛋白： |
质粒简介
pK7GWIWG2(I)是一个植物表达质粒。
质粒图谱
质粒序列
LOCUS Exported 13599 bp ds-DNA circular SYN 18-JUN-2017
DEFINITION synthetic circular DNA
KEYWORDS pK7GWIWG2(I)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13599)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..13599
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 28..253
/label=35s Terminator
protein_bind 263..387
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter 412..442
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
CDS 496..1155
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 1497..1802
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLL-SDK
VSRELYPVVHIGDESWRMMTTD-MASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(1843..1967)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
misc_feature complement(2035..2678)
/label=intron
protein_bind 2693..2817
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
CDS complement(2858..3163)
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLS-DK
VSRELYPVVHIGDESWRMMTTDM-ASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
CDS complement(3505..4164)
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
promoter complement(4218..4248)
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
protein_bind complement(4273..4397)
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter complement(4425..5460)
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
misc_feature 5482..5681
/label=RB
misc_feature 5543..5567
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 6867..7496
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 StaA
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 7930..8997
/codon_start=1
/product="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 RepA
/translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
MRYRNLIEGEASAGS"
rep_origin 9063..9257
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 9601..9741
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(9927..10515)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(10759..11550)
/codon_start=1
/gene="aadA"
/product="aminoglycoside adenylyltransferase (Murphy,
1985)"
/label=Spe
/note="confers resistance to spectinomycin and
streptomycin"
/translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
misc_feature 11840..12172
/label=LB
misc_feature 12078..12102
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
terminator 12195..12447
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(12487..13281)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(13315..13494)
/label=NOS promoter
/note="nopaline synthase promoter"
ORIGIN
1 ctagagggcc cgacgtcgca tgcctgcagg tcactggatt ttggttttag gaattagaaa
61 ttttattgat agaagtattt tacaaataca aatacatact aagggtttct tatatgctca
121 acacatgagc gaaaccctat aagaacccta attcccttat ctgggaacta ctcacacatt
181 attctggaga aaaatagaga gagatagatt tgtagagaga gactggtgat ttttgcggac
241 tctagcatgg ccgcgggata tcacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa
301 tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta cataatactg
361 taaaacacaa catatccagt cactatggcg gccgcattag gcaccccagg ctttacactt
421 tatgcttccg gctcgtataa tgtgtggatt ttgagttagg atccggcgag attttcagga
481 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa
541 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag
601 accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt
661 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg
721 gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc
781 catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag
841 tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct
901 aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt
961 tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa
1021 tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtc
1081 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg
1141 cagggcgggg cgtaaacgcg tggatccggc ttactaaaag ccagataaca gtatgcgtat
1201 ttgcgcgctg atttttgcgg tataagaata tatactgata tgtatacccg aagtatgtca
1261 aaaagaggtg tgctatgaag cagcgtatta cagtgacagt tgacagcgac agctatcagt
1321 tgctcaaggc atatatgatg tcaatatctc cggtctggta agcacaacca tgcagaatga
1381 agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat caggaaggga tggctgaggt
1441 cgcccggttt attgaaatga acggctcttt tgctgacgag aacagggact ggtgaaatgc
1501 agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga
1561 gtgatattat tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc
1621 tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc
1681 gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg
1741 atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat
1801 aaatgtcagg ctcccttata cacagccagt ctgcaggtcg accatagtga ctggatatgt
1861 tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata
1921 tttatatcat tttacgtttc tcgttcagct ttcttgtaca aagtggtgat atcactagtg
1981 cggccgcctg caggtcgacc atatggtcga cctgcaggcg gccgcactag tgatgctgtt
2041 atgttcagtg tcaagctgac ctgcaaacac gttaaatgct aagaagttag aatatatgag
2101 acacgttaac tggtatatga ataagctgta aataaccgag tataaactca ttaactaata
2161 tcacctctag agtataatat aatcaaattc gacaatttga ctttcaagag taggctaatg
2221 taaaatcttt atatatttct acaatgttca aagaaacagt tgcatctaaa cccctatggc
2281 catcaaattc aatgaacgct aagcttaata tgactctcaa taaagtctca taccaacaag
2341 tgccacctta ttcaaccatc aagaaaaaag ccaaaattta tgctactcta aggaaaactt
2401 cactaaagaa gacgatttag agtgttttac caagaatttc tgtcatctta ctaaacaact
2461 aaagatcggt gtgatacaaa acctaatctc attaaagttt atgctaaaat aagcataatt
2521 ttacccacta agcgtgacca gataaacata actcagcaca ccagagcata tatattggtg
2581 gctcaaatca tagaaactta cagtgaagac acagaaagcc gtaagaagag gcaagagtat
2641 gaaaccttac ctcatcattt ccatgaggtt gcttctgatc ccgcgggata tcaccacttt
2701 gtacaagaaa gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat
2761 tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt cactatggtc
2821 gacctgcaga ctggctgtgt ataagggagc ctgacattta tattccccag aacatcaggt
2881 taatggcgtt tttgatgtca ttttcgcggt ggctgagatc agccacttct tccccgataa
2941 cggagaccgg cacactggcc atatcggtgg tcatcatgcg ccagctttca tccccgatat
3001 gcaccaccgg gtaaagttca cgggagactt tatctgacag cagacgtgca ctggccaggg
3061 ggatcaccat ccgtcgcccg ggcgtgtcaa taatatcact ctgtacatcc acaaacagac
3121 gataacggct ctctctttta taggtgtaaa ccttaaactg catttcacca gtccctgttc
3181 tcgtcagcaa aagagccgtt catttcaata aaccgggcga cctcagccat cccttcctga
3241 ttttccgctt tccagcgttc ggcacgcaga cgacgggctt cattctgcat ggttgtgctt
3301 accagaccgg agatattgac atcatatatg ccttgagcaa ctgatagctg tcgctgtcaa
3361 ctgtcactgt aatacgctgc ttcatagcac acctcttttt gacatacttc gggtatacat
3421 atcagtatat attcttatac cgcaaaaatc agcgcgcaaa tacgcatact gttatctggc
3481 ttttagtaag ccggatccac gcgtttacgc cccgccctgc cactcatcgc agtactgttg
3541 taattcatta agcattctgc cgacatggaa gccatcacag acggcatgat gaacctgaat
3601 cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg
3661 ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg
3721 attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc
3781 accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta
3841 ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg
3901 aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc
3961 attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt
4021 tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc
4081 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
4141 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgccggatc
4201 ctaactcaaa atccacacat tatacgagcc ggaagcataa agtgtaaagc ctggggtgcc
4261 taatgcggcc gccatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt
4321 ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcagct
4381 tttttgtaca aacttgtgat atcactagtg cggccgcctg caggtcgact agaatagtaa
4441 attgtaatgt tgtttgttgt ttgttttgtt gtggtaattg ttgtaaaaat acggatcgtc
4501 ctgcagtcct ctccaaatga aatgaacttc cttatataga ggaagggtct tgcgaaggat
4561 agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg
4621 aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt
4681 tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc
4741 atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc
4801 aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt
4861 ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca
4921 tgttgacgaa gattttcttc ttgtcattga gtcgtaaaag actctgtatg aactgttcgc
4981 cagtcttcac ggcgagttct gttagatcct cgatctgaat ttttgactcc atggcctttg
5041 attcagtagg aactactttc ttagagactc caatctctat tacttgcctt ggtttatgaa
5101 gcaagccttg aatcgtccat actggaatag tacttctgat cttgagaaat atatctttct
5161 ctgtgttctt gatgcagtta gtcctgaatc ttttgactgc atctttaacc ttcttgggaa
5221 ggtatttgat ctcctggaga ttattactcg ggtagatcgt cttgatgaga cctgccgcgt
5281 aggcctctct aaccatctgt gggtcagcat tctttctgaa attgaagagg ctaatcttct
5341 cattatcggt ggtgaacatg gtatcgtcac cttctccgtc gaactttctt cctagatcgt
5401 agagatagag aaagtcgtcc atggtgatct ccggggcaaa ggagatcagc ttggctctag
5461 tcgaccatat gggagagctc aagcttagct tgagcttgga tcagattgtc gtttcccgcc
5521 ttcagtttaa actatcagtg tttgacagga tatattggcg ggtaaaccta agagaaaaga
5581 gcgtttatta gaataacgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt
5641 tgtatgtgca tgccaaccac agggttcccc tcgggatcaa agtactttga tccaacccct
5701 ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag ccgtcttctg aaaacgacat
5761 gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg cccttttcct ggcgttttct
5821 tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac tagaaccgga gacattacgc
5881 catgaacaag agcgccgccg ctggcctgct gggctatgcc cgcgtcagca ccgacgacca
5941 ggacttgacc aaccaacggg ccgaactgca cgcggccggc tgcaccaagc tgttttccga
6001 gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc aggatgcttg accacctacg
6061 ccctggcgac gttgtgacag tgaccaggct agaccgcctg gcccgcagca cccgcgacct
6121 actggacatt gccgagcgca tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc
6181 gtgggccgac accaccacgc cggccggccg catggtgttg accgtgttcg ccggcattgc
6241 cgagttcgag cgttccctaa tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc
6301 ccgaggcgtg aagtttggcc cccgccctac cctcaccccg gcacagatcg cgcacgcccg
6361 cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca
6421 tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa gtgacgccca ccgaggccag
6481 gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc gacgccctgg cggccgccga
6541 gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg acggccagga cgaaccgttt
6601 ttcattaccg aagagatcga ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc
6661 gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg gtttgtctga tgccaagctg
6721 gcggcctggc cggccagctt ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga
6781 tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta
6841 aataaacaaa tacgcaaggg gaacgcatga aggttatcgc tgtacttaac cagaaaggcg
6901 ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc cctgcaactc gccggggccg
6961 atgttctgtt agtcgattcc gatccccagg gcagtgcccg cgattgggcg gccgtgcggg
7021 aagatcaacc gctaaccgtt gtcggcatcg accgcccgac gattgaccgc gacgtgaagg
7081 ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc ccaggcggcg gacttggctg
7141 tgtccgcgat caaggcagcc gacttcgtgc tgattccggt gcagccaagc ccttacgaca
7201 tatgggccac cgccgacctg gtggagctgg ttaagcagcg cattgaggtc acggatggaa
7261 ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg
7321 ttgccgaggc gctggccggg tacgagctgc ccattcttga gtcccgtatc acgcagcgcg
7381 tgagctaccc aggcactgcc gccgccggca caaccgttct tgaatcagaa cccgagggcg
7441 acgctgcccg cgaggtccag gcgctggccg ctgaaattaa atcaaaactc atttgagtta
7501 atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc
7561 acgcagcagc aaggctgcaa cgttggccag cctggcagac acgccagcca tgaagcgggt
7621 caactttcag ttgccggcgg aggatcacac caagctgaag atgtacgcgg tacgccaagg
7681 caagaccatt accgagctgc tatctgaata catcgcgcag ctaccagagt aaatgagcaa
7741 atgaataaat gagtagatga attttagcgg ctaaaggagg cggcatggaa aatcaagaac
7801 aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg aacgggcggt tggccaggcg
7861 taagcggctg ggttgtctgc cggccctgca atggcactgg aacccccaag cccgaggaat
7921 cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg gcgcggcgct gggtgatgac
7981 ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca
8041 cgccccggtg aatcgtggca agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg
8101 ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg gcgacgagca accagatttt
8161 ttcgttccga tgctctatga cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc
8221 gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg tgatccgcta cgagcttcca
8281 gacgggcacg tagaggtttc cgcagggccg gccggcatgg ccagtgtgtg ggattacgac
8341 ctggtactga tggcggtttc ccatctaacc gaatccatga accgataccg ggaagggaag
8401 ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg acgtactcaa gttctgccgg
8461 cgagccgatg gcggaaagca gaaagacgac ctggtagaaa cctgcattcg gttaaacacc
8521 acgcacgttg ccatgcagcg tacgaagaag gccaagaacg gccgcctggt gacggtatcc
8581 gagggtgaag ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag
8641 tacatcgaga tcgagctagc tgattggatg taccgcgaga tcacagaagg caagaacccg
8701 gacgtgctga cggttcaccc cgattacttt ttgatcgatc ccggcatcgg ccgttttctc
8761 taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca gatggttgtt caagacgatc
8821 tacgaacgca gtggcagcgc cggagagttc aagaagttct gtttcaccgt gcgcaagctg
8881 atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg aggcggggca ggctggcccg
8941 atcctagtca tgcgctaccg caacctgatc gagggcgaag catccgccgg ttcctaatgt
9001 acggagcaga tgctagggca aattgcccta gcaggggaaa aaggtcgaaa aggtctcttt
9061 cctgtggata gcacgtacat tgggaaccca aagccgtaca ttgggaaccg gaacccgtac
9121 attgggaacc caaagccgta cattgggaac cggtcacaca tgtaagtgac tgatataaaa
9181 gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc
9241 cgcctggcct gtgcataact gtctggccag cgcacagccg aagagctgca aaaagcgcct
9301 acccttcggt cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat cgcggccgct
9361 ggccgctcaa aaatggctgg cctacggcca ggcaatctac cagggcgcgg acaagccgcg
9421 ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga
9481 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
9541 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
9601 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
9661 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
9721 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
9781 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
9841 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
9901 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
9961 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
10021 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
10081 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
10141 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
10201 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
10261 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
10321 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
10381 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
10441 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
10501 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
10561 gaaaactcac gttaagggat tttggtcatg catgatatat ctcccaattt gtgtagggct
10621 tattatgcac gcttaaaaat aataaaagca gacttgacct gatagtttgg ctgtgagcaa
10681 ttatgtgctt agtgcatcta atcgcttgag ttaacgccgg cgaagcggcg tcggcttgaa
10741 cgaatttcta gctagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg
10801 gacaaattct tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata
10861 agcctgtcta gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca
10921 gtcggcagcg acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga
10981 caacgtaagc actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt
11041 taaggtttca tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc
11101 cgccgctgga cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag
11161 atcaatgtcg atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc
11221 tccaaattgc agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac
11281 aatggtgact tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa
11341 aaggtcgttg atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag
11401 caaatcaata tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac
11461 ggccagcaac gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt
11521 cgatacttcg gcgatcaccg cttcccccat gatgtttaac tttgttttag ggcgactgcc
11581 ctgctgcgta acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc
11641 ttgctgcttg gatgcccgag gcatagactg taccccaaaa aaacatgtca taacaagaag
11701 ccatgaaaac cgccactgcg ccgttaccac cgctgcgttc ggtcaaggtt ctggaccagt
11761 tgcgtgacgg cagttacgct acttgcatta cagcttacga accgaacgag gcttatgtcc
11821 actgggttcg tgcccgaatt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca
11881 tgctaccctc cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg
11941 aatagcatcg gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg
12001 tcccggactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg
12061 agctgttggc tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa
12121 cttaataaca cattgcggac gtttttaatg tactgaatta acgccgaatt gaattatcag
12181 cttgcatgcc ggtcgatcta gtaacataga tgacaccgcg cgcgataatt tatcctagtt
12241 tgcgcgctat attttgtttt ctatcgcgta ttaaatgtat aattgcggga ctctaatcat
12301 aaaaacccat ctcataaata acgtcatgca ttacatgtta attattacat gcttaacgta
12361 attcaacaga aattatatga taatcatcgc aagaccggca acaggattca atcttaagaa
12421 actttattgc caaatgtttg aacgatctgc ttgactctag ctagagtccg aaccccagag
12481 tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc
12541 ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat
12601 atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc
12661 gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccgtg
12721 ggtcacgacg agatcctcgc cgtcgggcat ccgcgccttg agcctggcga acagttcggc
12781 tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat
12841 ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg
12901 atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc
12961 aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc
13021 cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga
13081 tagccgcgct gcctcgtctt ggagttcatt cagggcaccg gacaggtcgg tcttgacaaa
13141 aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt
13201 ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg
13261 caatccatct tgttcaatca tgcctcgatc gagttgagag tgaatatgag actctaattg
13321 gataccgagg ggaatttatg gaacgtcagt ggagcatttt tgacaagaaa tatttgctag
13381 ctgatagtga ccttaggcga cttttgaacg cgcaataatg gtttctgacg tatgtgctta
13441 gctcattaaa ctccagaaac ccgcggctga gtggctcctt caacgttgcg gttctgtcag
13501 ttccaaacgt aaaacggctt gtcccgcgtc atcggcgggg gtcataacgt gactccctta
13561 attctcatgt atgataattc gagggtaccc ggggatcct
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
| P3074/pENTR223-DBR1（1点突变1同义突变）人源基因质粒 |
| P3078/pENTR223-IP6K2人源基因质粒 |
| P3079/pENTR223-ENY2人源基因质粒 |
| P3080/pENTR223-HIST2H4A人源基因质粒 |
| P3081/pENTR223-MANBAL人源基因质粒 |
| P3082/pENTR223-C16orf57人源基因质粒 |
| P3083/pENTR223-ACBD7(一同义突变）人源基因质粒 |
| P3084/pENTR223-GNG4人源基因质粒 |
| P3085/pENTR223-FXYD7人源基因质粒 |
| P3086/pENTR223-CTNNBIP1人源基因质粒 |
| P3087/pENTR223-RPL39L人源基因质粒 |
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pK7GWIWG2(I)植物表达质粒
￥100 - 1000
